Brucellosis is an infectious disease caused by bacteria of the genus Brucella. There are various Brucella species that are capable of infecting both wildlife and livestock. The principal cause of brucellosis in cattle is the bacterium B. abortus. Infected cattle commonly have high incidences of spontaneous abortions, arthritic joints, and retained placenta following calving. In the United States, infected cows are often killed. Sheep and goats are the preferred hosts of B. melitensis, which is the Brucella species most virulent for humans. Humans can become infected by coming in contact with infected animals or animal products, such as unpasteurized milk, that are contaminated with these bacteria.
Vaccines are used to protect against diseases, which are caused by pathogens. These pathogens are microbial organisms, such as bacteria and viruses, which affect animals, including humans. Vaccines are primarily derived from a pathogen by producing and administering either: a) an attenuated or avirulent version of the pathogen; b) the killed pathogen; c) extracted protective antigens or antigen mixes of the pathogen (homologous antigens); or d) a micro-organism expressing one or more protective antigens encoded by cloned genes originating in a microbial pathogen different from the vaccine strain (heterologous antigens).
Vaccines for both bacteria and viruses are engineered from microorganisms expressing one or more protective antigens, as described by K. Jones and M. Sheppard in Designer Vaccines, CRC Press (1997). Vaccines are intended to produce an immune response in the recipient consisting of at least one of an antibody mediated or T cell mediated immune response, thereby preventing future infection by a pathogen, or fighting a current pathogenic infection. In particular, vaccines against facultative intracellular pathogens, those growing inside the cells of the infected host, need to induce a strong and appropriate cell mediated immune response. In contrast, vaccines against obligate extracellular pathogens need to induce an appropriate antibody mediated immune response. Often, regardless of the pathogen, an appropriate combined antibody and cellular mediated immune response leads to sufficient protection or relief from infection. In order to achieve this protection or relief from infection, vaccines may express one or more homologous antigens, heterologous antigens, or a combination of both.
Vaccines may be administered to vertebrates both to prevent and treat infection by pathogens. Thus, vaccines are frequently administered to prevent the spread of a disease caused by a pathogen. In particular, herd animals, such as cows, goats, sheep and swine, are often vaccinated to prevent the spread of a disease among members of the herd. Further, because certain diseases may travel between vertebrates, including travel between various animals and between animals and humans, vaccines are used to prevent the spread of disease between various species, usually by administration to the infected animal and other uninfected animals in the immediate vicinity. However, other animals in the area which are less likely to contract the disease may also be vaccinated as a prophylactic measure. For example, an infected cow and its as yet uninfected herd may be vaccinated to treat a disease and prevent its further spread. As a prophylactic measure, other animals which are likely to contract the disease from the infected cow, such as neighboring cows, sheep or humans, may be vaccinated as well.
B. abortus strain RB51 is a stable rough mutant derived from the wild-type virulent strain B. abortus 2308 [1]. This strain is currently the official USDA approved vaccine against cattle brucellosis in the United States and elsewhere. The safety and protective efficacy of strain RB51 have been well demonstrated under laboratory and field conditions [2, 3]. Protection afforded by strain RB51 vaccination is due to induction of cell-mediated immune mechanisms including antigen-specific induction of IFN-γ production [4-6]. Multiple studies have exploited the advantageous vaccinal qualities of strain RB51 as a host for the delivery of protective antigens of other intracellular pathogens in which a Th1 type immune response or cell-mediated immunity is essential for protection [7, 8].
Previously, plasmid-based expression systems have been utilized to express heterologous proteins in strain RB51 [5, 8-13]. However, these expression vectors usually encode antibiotic resistance markers. Moreover, the U.S. Food and Drug Administration strongly discourages and strictly regulates the introduction of antibiotic resistance genes into live attenuated vaccines [14]. In addition, often such expression systems are unstable in the absence of antibiotic selection pressure. To overcome this hurdle, an environmentally safe leucine auxotroph strain of RB51 (RB51leuB) was created to over-express foreign antigens without using antibiotic resistance marked plasmids [15]. The production of this strain is described in US 2010/0226942 herein incorporated by reference. This auxotrophic strain can be complemented with the pNS4 vector expressing the wild-type leuB gene. This complementation allows for survival of the strain in leucine-deficient minimal medium and under nutrient-limiting conditions in vivo, thus providing selective pressure for plasmid maintenance without using antibiotic selection markers [16].
Although the creation of the strain RB51leuB and pNS4 has led to a safer and more stable platform for expression of other foreign antigens, additional modifications are necessary to allow for better and more consistent expression in this host-vector system. Additionally, a need remains in the art for immunogenic compositions and multivalent vaccines to combat Brucella and other bacterial infections such as those caused by mycobacteria. Similar to Brucella, humans can become infected with M. tuberculosis by coming in contact with infected animals or animal products, such as unpasteurized milk, that are contaminated with these bacteria. Additionally, the available Brucella vaccines can be effective in controlling brucellosis, but they can have numerous drawbacks, such as interference with diagnostic tests, pathogenicity for humans, and potential to cause abortion in pregnant animals.